The present invention relates to a method for determining the activity of the angiotensin-converting enzyme, and more particularly to a method for determining the activity of the angiotensin-converting enzyme comprising the steps of mixing a liquid containing (i) a synthetic substrate of X-hippuryl-L-dipeptide having the formula ##STR3## wherein X is OH, NH.sub.2 or N(CH.sub.3).sub.2, and wherein the pair of substituents (R.sub.1, R.sub.2) is either (a). (H,H), or ##STR4## (ii) hippuricase and (iii) 4-aminoantipyrine, with a liquid containing angiotensin converting enzyme, and measuring colorimetrically the concentration of a quinonimine dye which is formed by adding an oxidizing agent to the above mixture.
The angiotensin-converting enzyme (hereinafter referred to as ACE) acts on angiotensin I in the human body and liberates a C-terminal dipeptide (i.e., L-histidyl-L-leucine) from angiotensin I, so that an active type angiotensin II that has the effect of increasing blood pressure is produced. ACE plays an important role in the human body in collaboration with a renin-angiotensin enzyme group or with a quinine-kallikrein enzyme group. Furthermore, by checking the level of the ACE in the blood, diagnosis of sarcoidosis can be performed. Therefore, the measurement of the enzyme activity of ACE is meaningful physiologically and clinically.
The following methods of measuring the enzyme activity of ACE have been proposed:
(1) a method employing a radioisotope, (2) a method employing a fluorescent material, and (3) a method employing liquid chromatography. All of these methods, however, require special equipment.
In addition to the above-mentioned methods, a method proposed by Cushman et al is known. In this method, the synthetic substrate hippuryl-L-histidyl-L-leucine is allowed to react with a test sample containing ACE, for instance, with serum or body fluid, for a predetermined period of time to produce hippuric acid, and then hydrochloric acid is added to the reaction mixture to terminate the reaction. The hippuric acid thus produced is extracted with ethyl acetate. Ethyl acetate is then evaporated from the extracted solution, leaving a dried solid. The dried solid is dissolved in distilled water and the absorbence of the solution is determined in the ultraviolet region, so that the concentration of hippuric acid is determined and, from the concentration of hippuric acid, the activity of ACE is calculated. This method entails complicated steps and, therefore, is not always practical.